A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.European Journal of Human Genetics advance online publication, 4 February 2015; doi:10.1038/ejhg.2014.283

Study of USH1 Splicing Variants through Minigenes and Transcript Analysis from Nasal Epithelial Cells

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital profound deafness, vestibular areflexia and prepubertal retinitis pigmentosa. The first purpose of this study was to determine the pathologic nature of eighteen USH1 putative splicing variants found in our series and their effect in the splicing process by minigene assays. These variants were selected according to bioinformatic analysis. The second aim was to analyze the USH1 transcripts, obtained from nasal epithelial cells samples of our patients, in order to corroborate the observed effect of mutations by minigenes in patient’s tissues. The last objective was to evaluate the nasal ciliary beat frequency in patients with USH1 and compare it with control subjects. In silico analysis were performed using four bioinformatic programs: NNSplice, Human Splicing Finder, NetGene2 and Spliceview. Afterward, minigenes based on the pSPL3 vector were used to investigate the implication of selected changes in the mRNA processing. To observe the effect of mutations in the patient’s tissues, RNA was extracted from nasal epithelial cells and RT-PCR analyses were performed. Four MYO7A (c.470G>A, c.1342_1343delAG, c.5856G>A and c.3652G>A), three CDH23 (c.2289+1G>A, c.6049G>A and c.8722+1delG) and one PCDH15 (c.3717+2dupTT) variants were observed to affect the splicing process by minigene assays and/or transcripts analysis obtained from nasal cells. Based on our results, minigenes are a good approach to determine the implication of identified variants in the mRNA processing, and the analysis of RNA obtained from nasal epithelial cells is an alternative method to discriminate neutral Usher variants from those with a pathogenic effect on the splicing process. In addition, we could observe that the nasal ciliated epithelium of USH1 patients shows a lower ciliary beat frequency than control subjects

La température module les profils d'acides gras chez les diatomées et l'impact d'herbicides

International audienceFatty acids are essential elements for the structure of biological membranes and for the storage of metabolic energy. They are used as a source of energy by metabolism at each trophic level, making fatty acids biochemically and physiologically important compounds (Neves et al. 2015). In the trophic chain, many fatty acids are only synthesized by microalgae and bacteria before being transferred via herbivorous invertebrates to fish and ultimately to humans (Arts et al. 2001). For example, highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid (EPA; C20:5n3), cannot be synthesized de novo or in insufficient proportions by animals (Saito and Aono 2014). That is why fatty acid analysis is commonly used to study trophic interactions in food chains. Generally, microalgae with a high proportion of EPA, such as diatoms, are an excellent source of food for animals but the concentrations of these different fatty acids can vary according to different environmental parameters including temperature, and may be affected by pesticide exposure (Brett et al. 2006, Robert et al. 2007, Burns et al. 2011, Li et al. 2014, Filimonova et al. 2016). The aim of this study is to investigate the impact of two herbicides combined to a 3°C-temperature increase on diatom's fatty acids and photosynthesis. To address this issue, two model freshwater diatoms (Gomphonema gracile and Nitzschia palea) were exposed to two herbicides, with two different cellular targets, at high concentration (diuron and s-metolachlor, C=10 µg/L) and two temperatures (T1=17,5 °C and T2=20,5°C). After a 1-week exposure, the analysis of photosynthesis was carried out by PhytoPAM measurements, and fatty acid composition of diatom cultures was determined by gas chromatography. For the control samples, the photosynthesis increased with the temperature for the both species mainly for Gomphonema gracile. Concerning the fatty acids composition of the both species, saturated and monounsaturated fatty acids decreased with temperature while polyunsaturated fatty acids increased. The 3°C increase in temperature enhanced the toxicity of diuron towards the photosynthesis of both species, while it reduced the effect of S-metolachlor for Gomphonema gracile. Fatty acids analysis are still ongoing. Our results highlight contrasting effects of a moderate increase of temperature on the toxicity of pesticides towards diatoms, at the physiological (photosynthesis) and structural (fatty acid content) levels

Impact de trois pesticides sur les acides gras de Gomphonema gracile

International audienceFatty acids are essential elements for the structure of biological membranes and for the storage of metabolic energy. They are used as a source of energy by metabolism at each trophic level, making fatty acids biochemically and physiologically important compounds (Neves et al. 2015). In the trophic chain, many fatty acids are only synthesized by microalgae and bacteria before being transferred via herbivorous invertebrates to fish and ultimately to humans (Arts et al. 2001). For example, highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid (EPA; C20:5n3), can not be synthesized de novo or in insufficient proportions by animals (Saito and Aono 2014). That is why fatty acid analysis is commonly used to study trophic interactions in food chains. Generally, microalgae with a high proportion of EPA, such as diatoms, are an excellent source of food for animals but the concentrations of these different fatty acids can vary according to the stage of growth of the organism and according to different environmental parameters including pesticide exposure (Brett et al. 2006, Robert et al. 2007, Burns et al. 2011, Filimonova et al. 2016). Moreover, for several years, the intensive use of pesticides caused many problems to the environment, making pesticides major pollutants of aquatic ecosystems (Aydinalp and Porca 2004). The aim of this study is to investigate the impact of 3 pesticides on diatom's fatty acids. To address this issue, a model freshwater diatom (Gomphonema gracile) was exposed to three herbicides, with three different cellular targets, at environmentally relevant and higher concentrations (diuron and S-metolachlor, C1= 1 µg/L and C2= 10 µg/L; glyphosate, C1= 5 µg/L and C2= 50 µg/L). After a 1-week exposure, fatty acid compositions of diatoms were determined by gas chromatography. In comparison with control samples the percentage of 1) polyunsaturated fatty acids (PUFA) decreased with S-metolachlor contamination (C2); 2) saturated fatty acid (SFA) and monounsaturated (MUFA) decreased with diuron and glyphosate exposure (C2). The decrease of PUFA is a direct impact and can be explained by the mode of action of S-metolachlor which inhibits elongases. Concerning diuron and glyphosate, the decrease of SFA and MUFA can reflect an indirect effect, which can be explained by the mode of action of these two pesticides which respectively blocks electron transfer in photosynthesis, and inhibits the synthesis of aromatic aminoacids

Impacts de trois pesticides sur les acides gras de Gomphonema gracile

International audienceFatty acids are essential elements for the structure of biological membranes and for the storage of metabolic energy. They are used as a source of energy by metabolism at each trophic level, making fatty acids biochemically and physiologically important compounds (Neves et al. 2015). In the trophic chain, many fatty acids are only synthesized by microalgae and bacteria before being transferred via herbivorous invertebrates to fish and ultimately to humans (Arts et al. 2001). For example, highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid (EPA; C20:5n3), can not be synthesized de novo or in insufficient proportions by animals (Saito and Aono 2014). That is why fatty acid analysis is commonly used to study trophic interactions in food chains. Generally, microalgae with a high proportion of EPA, such as diatoms, are an excellent source of food for animals but the concentrations of these different fatty acids can vary according to the stage of growth of the organism and according to different environmental parameters including pesticide exposure (Brett et al. 2006, Robert et al. 2007, Burns et al. 2011, Filimonova et al. 2016). Moreover, for several years, the intensive use of pesticides caused many problems to the environment, making pesticides major pollutants of aquatic ecosystems (Aydinalp and Porca 2004). The aim of this study is to investigate the impact of 3 pesticides on diatom's fatty acids. To address this issue, a model freshwater diatom (Gomphonema gracile) was exposed to three herbicides, with three different cellular targets, at environmentally relevant and higher concentrations (diuron and S-metolachlor, C1= 1 µg/L and C2= 10 µg/L; glyphosate, C1= 5 µg/L and C2= 50 µg/L). After a 1-week exposure, fatty acid compositions of diatoms were determined by gas chromatography. In comparison with control samples the percentage of 1) polyunsaturated fatty acids (PUFA) decreased with S-metolachlor contamination (C2); 2) saturated fatty acid (SFA) and monounsaturated (MUFA) decreased with diuron and glyphosate exposure (C2). The decrease of PUFA is a direct impact and can be explained by the mode of action of S-metolachlor which inhibits elongases. Concerning diuron and glyphosate, the decrease of SFA and MUFA can reflect an indirect effect, which can be explained by the mode of action of these two pesticides which respectively blocks electron transfer in photosynthesis, and inhibits the synthesis of aromatic aminoacids

Impact de 3 pesticides sur les acides gras de la diatomée d'eau douce Gomphonema gracile

International audienceDepuis plusieurs années, l'usage intensif de pesticides a causé de nombreux problèmes à l'environnement, faisant des pesticides des polluants majeurs des écosystèmes aquatiques (Aydinalp & Porca 2004). Parmi les indicateurs biologiques, les biomarqueurs doivent être développés en raison de leur capacité à révéler précocement des effets toxiques sur les organismes vivants. Le but de cette étude est d'évaluer les spécificités/sensibilités des profils d'acides gras (composition en acides gras saturés, insaturés et polyinsaturés par chromatographie gazeuse couplée à un détecteur à ionisation de flamme) des diatomées en comparaison avec des descripteurs d'effet « classiques » (inhibition de la photosynthèse, croissance, analyses génétiques). Des cultures de la diatomée d'eau douce Gomphonema gracile sont soumises séparément à 3 pesticides ayant des cibles cellulaires différentes, à une concentration environnementale et une concentration plus élevée (diuron et Smétolachlore, C1= 1 µg/L et C2= 10 µg/L ; glyphosate, C1= 5 µg/L and C2= 50 µg/L). Les diatomées sont exposées durant leur phase de croissance exponentielle à une température et une intensité lumineuse respectivement de 17°C et 67 µmol.m-2.s-1. Après une semaine d'exposition, la composition des diatomées en acides gras est déterminée en parallèle des descripteurs « classiques ». Tandis que les effets des pesticides sur les descripteurs « classiques » sont pas ou peu sensibles, notamment aux plus faibles concentrations d'exposition, certains profils d'acides gras semblent être spécifiques à la nature de la contamination chimique. A l'heure actuelle des analyses approfondies des acides gras sont encore en cours. Finalement, cette étude fournit une base pour l'utilisation de la composition en acides gras des diatomées comme biomarqueur de la contamination toxique des eaux douces

Impact du diuron et du S-métolachlore sur la diatomée d'eau douce Gomphonema gracile : Complémentarité entre profils d'acides gras et différents types d'indicateurs écotoxicologiques

International audienceFatty acids (FA) are crucial for the maintenance of membrane fluidity and play a central role in metabolic energy storage. Polyunsaturated fatty acids play an essential ecological role since they are key parameters in the nutritional value of algae. Pesticide impacts on fatty acid profiles have been documented in marine microalgae, but remain understudied in freshwater diatoms. The aims of this study were to: 1) investigate the impact of diuron and S-metolachlor on "classical descriptors" (photosynthesis, growth rate, pigment contents, and on the expression levels of target genes in freshwater diatoms), 2) examine the impact of these pesticides on diatom fatty acid profiles and finally, 3) compare fatty acid profiles and "classical descriptor" responses in order to evaluate their complementarity and ecological role. To address this issue, the model freshwater diatom Gomphonema gracile was exposed during seven days to diuron and S-metolachlor at 10 µg.L

L'indice Oméga 3 des végétaux comme indicateurs de performances de procédés de traitement

International audienceThe Omega-3 Index of macrophytes to improve the assessment of the treatment performance of constructed wetlands receiving treated wastewater

Variabilité interspécifique des profils d'acides gras de diatomées d'eau douce en réponse aux herbicides

International audienceMany studies showed the impact of pesticides on diatom's metabolism. Diuron decreases the photosynthetic activity and synthesis of polyunsaturated fatty acid whereas s-metolachlor decreases the synthesis of long chain fatty acids. Fatty acids (FA) are essential elements for the structure of biological membranes and for the storage of metabolic energy. For example polyunsaturated fatty acids (PUFA) play a key role in membrane fluidity. Various studies demonstrated impacts of chemical contamination on fatty acid profiles. Furthermore, variability of fatty acid profiles of marine microalga in response to herbicides has been proved. However, to our knowledge scarce studies showed the interspecific variability of fatty acid profiles of freshwater diatoms especially in response to a pesticide contamination. The aims of this study were to investigate the interspecific variability of two freshwater diatoms on photosynthesis, growth rate, concentration in chlorophyll a and carotene and induction or repression of interest genes; Study this variability on fatty acid profiles; with and without pesticides. To address this issue, two model freshwater diatoms, Gomphonema gracile (GGRA) and Nitzschia palea (NPAL), were exposed separately during seven days to diuron and s-metolachlor at high concentration (C=10 µg/L) and at 20.5°C. For control condition, NPAL had a higher chlorophyll a and carotenoid concentration than GGRA. An interspecific variability of fatty acid profiles was observed with a lower percentage of saturated fatty acid (SFA) and highly unsaturated fatty acid (HUFA) for NPAL than GGRA. However, NPAL had a higher percentage of 22:6n3 than GGRA. Diuron decreased the percentage of chlorophyll a and carotenoid concentration of NPAL while no significant effect was observed for GGRA. Diuron increased percentage of SFA for NPAL whereas a decrease was noted for GGRA; the percentage of 14:0 and 16:0 increased for NPAL while it decreased for GGRA. NPAL had a different fatty acid profile compared to GGRA and seemed to have diverging mechanisms of protection in response to a contamination. This study proved the importance to taking into account interspecific variability of fatty acid profiles of diatoms for further use them as biomarkers of toxic contamination in fresh waters

Devenir des micropolluants et bioindicateur lipidique en Zone de rejet végétalisée

National audienc